Inhibiting the proteasome reduces molecular and biological impacts of the natural product insecticide, spinosad

Abstract

BACKGROUND Insecticide targets are often identified by mutations that confer resistance, but the intricacies of insecticide binding and downstream processes leading to insect death often remain obscure. Mutations in α6‐like nicotinic acetylcholine receptor subunit genes have been associated with high levels of resistance to spinosad in many insect species, including Drosophila melanogaster. Here, we aimed to expand our understanding of the effects of the natural product insecticide spinosad on its protein target, the α6 subunit, using genetic tools available in D. melanogaster. RESULTS Functional, fluorescently tagged Dα6 subunits (Dα6YFP) were developed to allow observation of the protein in vivo. Larvae expressing Dα6YFP were exposed to a sub‐lethal concentration of spinosyn A (0.025 ppm) for 6 days, leading to a 64% reduction in fluorescence relative to unexposed larvae. Direct application of high doses of spinosyn A to dissected larval brains resulted in a visible 38.25% decrease in Dα6YFP within 20 min, indicating that degradation of the Dα6 protein occurred in response to spinosyn A exposure. Chemical inhibition of the proteasome system using the multiple myeloma treatment drug, PS‐341 reduced loss of Dα6YFP in response to spinosyn A at the 20‐min time point to 6.35%. In addition, in vivo administration of PS‐341 prior to spinosad exposure reduced the effect of spinosad on larval activity. CONCLUSION Based on these data, we propose that exposure to spinosad leads to degradation of the α6‐like target protein, a potentially novel element in the mode of action of spinosyns that may contribute to their toxicity towards insects. © 2021 Society of Chemical Industry

Low doses of the organic insecticide spinosad trigger lysosomal defects, ROS driven lipid dysregulation and neurodegeneration in flies

“…No increase in the area occupied by lysosomes was observed after exposure to imidacloprid, showing that this is a spinosad specific response ( Figure 2 – figure supplement 1 ). These observations, in combination with the findings of Nguyen et al (2021) suggested that binding of spinosad to Dα6 nAChRs may promote their trafficking to lysosomes. To investigate this hypothesis, the brains of larvae expressing a fluorescently (CFP) tagged Dα6 nAChR subunit were stained with LysoTracker.…”

“…Blocked neuronal receptors can be recycled from the plasma membrane through endocytosis (Saheki and De Camilli, 2012). Our data indicate that spinosad exposure leads to the removal of Dα6 nAChRs from neuronal membranes (Nguyen et al, 2021) and localization to enlarged lysosomes, resulting in lysosomal expansion ( Figure 2C ) and lysosomal dysfunction.…”

“…Recycling defects in neuronal cells caused by LSD impair cell function, ultimately triggering neurodegeneration (Darios and Stevanin, 2020). Nguyen et al (2021) recently showed that flies treated with a proteasome inhibitor drug, bortezomib, present with a reduced loss of Dα6 from neuronal membranes when exposed to spinosad. That suggests that the proteasome degradation pathway could also be involved in recycling spinosad-blocked Dα6 subunits.…”

“…It is currently accepted that spinosad causes an increased sensitivity to ACh in certain nAChRs and an enhanced response at some GABAergic synapses, causing involuntary muscle contractions, paralysis and death (Biondi et al, 2012; Perry et al, 2011; Salgado, 1998). A recent study (Nguyen et al, 2021) showed that both acute and chronic exposures to spinosad causes Dα6 protein levels in the larval brain to decrease. A rapid loss of Dα6 protein during acute exposure was blocked by inhibiting the proteasome system (Nguyen et al, 2021).…”

“…A recent study (Nguyen et al, 2021) showed that both acute and chronic exposures to spinosad causes Dα6 protein levels in the larval brain to decrease. A rapid loss of Dα6 protein during acute exposure was blocked by inhibiting the proteasome system (Nguyen et al, 2021). As Dα6 loss of function mutants are viable (Perry et al, 2007; Perry et al 2021), it was suggested that the toxicity of spinosad may be due to overloading of protein degradation pathways and/or the internalisation of spinosad where it may cause cellular damage.…”